Structures of New Phenolics Isolated from Licorice, and the Effectiveness of Licorice Phenolics on Vancomycin-Resistant Enterococci

Licorice, which is the underground part of Glycyrrhiza species, has been used widely in Asian and Western countries as a traditional medicine and as a food additive. Our continuous investigation on the constituents of roots and stolons of Glycyrrhiza uralensis led to the isolation of two new phenolics, in addition to 14 known compounds. Structural studies including spectroscopic and simple chemical derivatizations revealed that both of the new compounds had 2-aryl-3-methylbenzofuran structures. An examination of the effectiveness of licorice phenolics obtained in this study on vancomycin-resistant strains Enterococcus faecium FN-1 and Enterococcus faecalis NCTC12201 revealed that licoricidin showed the most potent antibacterial effects against both of E. faecalis and E. faecium with a minimum inhibitory concentration (MIC) of 1.9 × 10−5 M. 8-(γ,γ-Dimethylallyl)-wighteone, isoangustone A, 3'-(γ,γ-dimethylallyl)-kievitone, glyasperin C, and one of the new 3-methyl-2-phenylbenzofuran named neoglycybenzofuran also showed potent anti-vancomycin-resistant Enterococci effects (MIC 1.9 × 10−5–4.5 × 10−5 M for E. faecium and E. faecalis). The HPLC condition for simultaneous detection of the phenolics in the extract was investigated to assess the quality control of the natural antibacterial resource, and quantitative estimation of several major phenolics in the extract with the established HPLC condition was also performed. The results showed individual contents of 0.08%–0.57% w/w of EtOAc extract for the major phenolics in the materials examined.


Introduction
Infectious diseases caused by multidrug-resistant bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) are serious problems worldwide [1]. Although Enterococcus bacteria are considered ordinary components in the healthy human intestinal flora, they are responsible for complicated urinary tract infections and serious endocarditis [2]. Enterococcus faecium and Enterococcus faecalis account for >95% of Enterococcus isolates from clinical cultures [3], and only a few drugs such as linezolid and a combination of quinupristin and dalfopristin are used clinically for VRE [4]. Since the adverse effects of these drugs have been revealed and drug resistance to them may appear soon, the development of a new group of low toxicity antibacterial agents is needed. Licorice has been used as a food sweetener and is one of the oldest and most frequently used crude drugs in traditional medicine, particularly in Asian countries. A variety of pharmaceutical functions, such as antiulcer, anti-inflammatory, antiviral, and anticarcinogenic activities have been reported for licorice constituents [5][6][7][8], and the antibacterial effects of licorice phenolics have been demosntrated for various bacterial species [9][10][11][12][13][14]. The effect of a compound isolated from licorice, gancaonin I (1), on VRE was also demonstrated in a previous study [15].
Our continuous studies have revealed the antibacterial effects of several licorice phenolics on MRSA, particularly those with both γ,γ-dimethylallyl (prenyl) and hydroxyl groups [16]. Licoricidin (2) has the same structural features and displays a suppressive effect on oxacillin resistance shown by MRSA [16]. We also reported the anti-VRE effects of several licorice phenolics in a previous study [17]. Our further investigations have led to the isolation of 16 phenolic compounds including two new compounds with rarely occurring 2-aryl-3-methylbenzofuran structures. This paper explains the structural determination of the new compounds and the effects of those phenolics on two VRE stains. In addition, an analytical condition for high-performance liquid chromatography (HPLC) to simultaneously analyze polyphenolic constituents in the EtOAc extract was established for quality control of the antibacterial resource, and several major phenolics in the extract were quantitated using the established HPLC condition.

Structures of the New Compounds
Compound A (8): This compound was obtained as a light brown powder. Its molecular formula was C 22  forming an ABX spin system, and a one-proton singlet at δ H 6.41 (H-5'). The spectrum also showed four sets of proton resonances at δ H 5.16 (1H, t, J = 6.6 H Z , H-2"), 3.32 (2H, d, J = 6.6 Hz, H-1"), 1.60 (3H, s), and 1.70 (3H, s) (2 × CH 3 at C-3"), which are assignable to those of a γ,γ-dimethylallyl (prenyl) group. In addition, proton resonances characteristic of two methoxyl groups at δ H 3.82 and 3.35 (3H each, s) and one methyl group at δ H 1.89 (3H, s) were seen in the aliphatic region of the spectrum.  9) and two methoxyl groups (δ C 60.7 and 55.0). In addition to these resonances, the spectrum showed a methyl carbon resonance (δ C 7.9), ascribable to the methyl group at C-3 of the 2-arylbenzofuran structure.
The assignments of these proton and carbon resonances were substantiated by the heteronuclear single quantum correlation (HSQC) and heteronuclear multiple-bond correlation (HMBC) spectral data as summarized in Table 1. Key HMBC correlations among them and the nuclear Overhauser effect spectroscopy (NOESY) correlations indicating the locations of the respective substituents on the 2-arylbenzofuran skeleton are shown in Figure 2.   The locations of the hydroxyl and methoxyl groups, and the prenyl group on the B-ring of this compound were shown by the following HMBC correlations. Correlation of the methylene proton resonance at δ H 3.32 (H-1") of the prenyl moiety and an -OCH 3 resonance (δ H 3.82) with a common oxygenated aromatic carbon resonance at δ C 160.0 (C-4') and also the correlations of the same methylene proton resonance (H-1") and a methoxyl proton resonance (δ H 3.35) with a common oxygenated carbon resonance at δ C 158.9 (C-2'). The carbon resonance at δ C 160.0 (C-4') was also correlated with the singlet proton resonance at δ H 6.41 (H-5', directly correlated with the C-5 carbon resonance at δ C 96.6 in the HSQC spectrum), and this proton resonance was also correlated with an oxygenated aromatic carbon at δ C 153.1 (C-6'), which was not correlated with any methoxyl proton resonance. The H-5' resonance was also correlated with the carbon resonances at δ C 114.2 (C-3') and δ C 102.1 (C-1') of the same aromatic ring. The last two carbon resonances were discriminated by a correlation of the methylene proton resonance (H-1") to the carbon resonance at δ C 114.2 (C-3'). The sequence C-1' (δ C 102.1)-C-2' (δ C 158.9, with a methoxyl group)-C-3' (δ C 114.2, with the prenyl group)-C-4' (δ C 160.0, with a methoxyl group)-C-5' (δ C 96.6)-C-6' (δ C 153.1) was thus assigned for the B-ring. In addition, the NOESY spectrum of this compound showed correlations of the methine proton resonance at δ H 5.16 (H-2" of the prenyl moiety) with the two methoxyl resonances at δ H 3.82 (-OCH 3 at C-4') and δ H 3.35 (-OCH 3 at C-2'), and the former methoxyl resonance also showed a correlation with the proton resonance at δ H 6.41 (C-5'), in agreement with the sequence described above.
The presence of a methyl group at C-3 was clearly indicated by the HMBC correlations of the methyl proton resonance at δ H 1.89 (H-10) with the carbon resonance at δ C 145.7 (C-2), 114.2 (C-3), and 123.0 (C-9). The NOESY correlations of this proton resonance with the methoxyl proton resonance at δ H 3. Hz)] resonances, forming an ABX system, indicated the location C-6 for the hydroxyl group. These data, and also the remaining HMBC correlations, satisfied the 2-aryl-3-methyl-6-hydroxybenzofuran structure.
Because structure 8 assigned to compound A was an analog of a compound reported previously, glycybenzofuran (16), the corresponding methylated products of compounds 8 and 16 were compared. As a result, product 8a from 8 was the same as that obtained by methylation of 16, as expected. The structure of compound A was thus substantiated to be 4'-O-methylglycybenzofuran (8).
Compound B (14): Compound B was obtained as a light brown powder. The molecular formula C 21 H 22 O 5 , which was the same as that of glycybenzofuran (16) The spectrum also showed a methyl carbon resonance at δ C 8.7, a methoxyl carbon resonance at δ C 60.6, and five carbon resonances due to a prenyl unit (δ C 17.8, 22.9, 25.5, 124.6, and 129.7).
The presence of the methyl group at C-3 was indicated by the HMBC correlations from the methyl proton resonance (H-10) at δ H 1.97 with C-2 (δ C 145.5), C-3 (δ C 114.4), and C-9 (δ C 123.8) and the NOESY correlations between the methyl resonance at δ H 1.97 and H-4 at δ H 7.23. The resonances of H-4, H-5, and H-7, forming an ABX system as shown by the 1 H-1 H COSY spectrum, indicated the location of a hydroxyl group at C-6, and the HMBC correlations ( Figure 2) concerning these aromatic proton resonances also satisfied the location C-6 of the hydroxyl group. Based on these findings, structure 14, which was isomeric to 16, was assigned to compound B which accordingly was named neoglycybenzofuran. Methylation of 14 afforded 8a and thus substantiated the structure 14 for neoglycybenzofuran.
The antibacterial effects of the EtOAc extract were comparable to those of potent anti-VRE constituents. Potential synergy and/or additive effects between the purified phenolics remain to be determined.

HPLC Analyses of Anti-VRE Phenolics for the Evaluation of EtOAc Extract from G. uralensis as a Source of Antibacterial Agent
Because of the important uses of licorice in traditional medicine, qualitative and quantitative analyses of licorice constituents and licorice products have been reported [31][32][33][34][35][36]. Remarkable anti-VRE effects of several licorice phenolics shown in our current and previous studies [17] suggested requirements of the identification and quantitation of those constituents. We therefore developed an HPLC-UV method for the simultaneous detection of major isolated phenolic constituents in EtOAc extract from G. uralensis, in order to evaluate the quality of the extract as a source of antibacterial agent.
The HPLC-UV profile of the EtOAc extract from Tohoku licorice used in the present study under the established condition is shown in Figure 3. Each constituent in the HPLC profile was identified by comparisons of its retention time, UV and MS spectra (data not shown) with those of the isolated one. The elution order of the identified constituents was as follows: demethylhomopterocarpan (10, t R 38.6 min), 7-O-methylluteone (3, t R 41.2 min), licopyranocoumarin (12, t R 46.0 min), glycybenzofuran (16, t R 46.6 min), glycyrol (18, t R 52.9 min), licoarylcoumarin (19, t R 68.0 min), licoriphenone (9,  Quantitative analysis of several compounds was performed under the same HPLC condition, and the amounts of the major phenolic constituents are shown in Table 3. Among these major phenolics, gancaonin I (1) and isoangustone A (6) showed potent anti-VRE effects. Table 3. Contents of major licorice phenolics in G. uralensis (Tohoku licorice) EtOAc extract.

Spectral Data
Compound A (4'-O-Methylglycybenzofuran, 8): This compound was obtained as a light brown powder; 1 H-and 13 C-NMR (see Table 1 Compound B (Neoglycybenzofuran, 14): This compound was obtained as a light brown powder; 1 H-and 13 C-NMR (see Table 1

Methylation of Compounds A and B, and Glycybenzofuran
Trimethylsilyldiazomethane solution (1 mL) was added to a solution of 8 (1 mg) in EtOH (0.1 mL), and the mixture was kept for 3 h at room temperature. After evaporating the solvent, the remaining product was purified by TLC on silica gel (Merck, silica gel F254) (CHCl 3 -MeOH, 15:1, v/v) to give a methyl derivative of compound A (8a). Detection was effected by UV absorption at 254 nm. 1

Antibacterial Assay
Estimations of the antibacterial effects of licorice phenolics on the VRE E. faecium FN-1 and E. faecalis NCTC 12201 used in this study were conducted using VRE kindly provided by Y. Ike, Gunma University. The bacterial cells were precultured in Mueller-Hinton broth at 37 °C under aerobic conditions. They were incubated in the presence of compounds with the concentrations obtained by serial two-fold dilution at 37 °C without shaking in the same broth for 24 h on microplates as shown in a previous paper [17], and their MICs were estimated as the lowest concentrations where the bacterial cells were not observed visually as reported previously [16,17], and were given based on triplicate experiments. DMSO was used for dissolving compounds hardly soluble in water, and the final concentrations were set at <1%, where DMSO has no effect. The positive control, linezolid, was dissolved in water.

Simultaneous HPLC Analysis of Phenolic Constituents in the EtOAc Extract of Licorice
Simultaneous analysis of licorice phenolics was carried out on an HPLC-DAD D-2000 HSM system, composed of an L-2130 pump (Hitachi, Tokyo, Japan) and an L-2455 DAD (Hitachi). The DAD was set for obtaining UV spectral data from 200 to 400 nm, and chromatograms at 280 nm were used for the quantitative analyses. The column used was an YMC-Pack pro C18 (6.0 mm i.d. × 150 mm) and was set in an oven at 40 °C. The mobile phase consisted of H 2 O/MeCN/MeCOOH (55:40:5, v/v/v), and the flow rate was set at 1.0 mL/min. Quantitation of 1, 6, 9, 18, 20, 21, 22, and 25 was based on the HPLC profile monitored at 280 nm.
Licorice (10 g) was pulverized and extracted with EtOAc (100 mL × 3). Approximately 10 mg of the dried extract powder was dissolved in 10 mL of MeOH and filtered with a 0.45 µm PTFE membrane filter prior to injection (8 μL of the filtrate at 1 mg/mL) was applied to HPLC analysis. Stock solutions of eight licorice phenolics (1, 6, 9, 18, 20, 21, 22 and 25) were prepared at 0.1 mg/mL in MeOH, and diluted in series (from 0.1 to 0.001 mg/mL) to produce eight individual standard curves, for which the correlation coefficients were determined between 0.991 and 0.999 under the described HPLC conditions.

Conclusions
Our present investigation on the EtOAc extract of G. uralensis led to the purification of 16 compounds. Among the compounds obtained, two new compounds, 8 and 14, had 2-aryl-3-methylbenzofuran structures, which rarely occur in Nature. The isolated phenolics were categorized into isoflavones (3, 6, 11, 15, and 17), isoflavans (2 and 13), isoflavanones (4, 5, and 7), a 3-arylcoumarin (12), a pterocarpan (10), 2-aryl-3-methylbenzofurans (8, 14, and 16), and a benzylphenylketone (9). As shown in our previous studies, licorice phenolics possess remarkable antibacterial effects against MRSA [16] and VRE [17]. The effects of the licorice phenolics isolated in the present study on VRE were examined. Based on their MIC values (Table 2), the antibacterial activities of the isoflavans and the isoflavones, bearing prenyl and phenolic hydroxyl groups, were promising. Our previous study [17] also indicated that compounds with prenyl moieties, such as gancaonin I (1), licoarylcoumarin (19), and glycycoumarin (21), showed noticeable anti-VRE effects. Taken together, we conclude that licorice phenolics, particularly those with prenyl moieties, could be used for the development of anti-VRE agents. The mechanisms of action of these phenolics as well as their potential synergistic effects remain to be clarified and the possibility of presence of potential synergistic effects between these identified licorice constituents are remained to be clarified. With regard to their promising antibiotic activities, the phenolic constituents from licorice could be used as lead compounds for developing new antibacterial agents.